DEV012054 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Different TCF genes have similar patterning activities in the neural tube. HH stage 11/12 embryos were electroporated with the indicated DNAs, and analysed 24 hours postelectroporation (PE) for progenitor gene expression. (A-F) Electroporation of TCF1^DN reduced expression of dorsal progenitor proteins Pax7 (A) and Pax6 (B). GFP was used as a reporter of transgene expression (C). Ventral genes Olig2 (D) and Nkx2.2 (E) were ectopically and cell-autonomously activated in electroporated GFP-expressing cells (F). (G-L) Electroporation of TCF4^DN, resulted in similar changes in progenitor gene expression. (M-R) Electroporation of TCF3 fused to the repressor domain of engrailed (TCF3^EnR) resulted in similar changes in progenitor gene expression. (S-U) Electroporation of TCF3 fused to the VP16 activator domain (TCF3^VP16) resulted in the opposite phenotype: the cell-autonomous reduction of ventral gene expression

• Supplemental Figure S2 -

  Fig. S2. Wnt patterning activity depends on Gli. (A) HH stage 11/12 embryos were electroporated with the indicated DNAs (see diagram), together with the Gli-BS reporter containing 8Gli-binding sites. Embryos were assayed 24 hours PE for luciferase activity. Gli3^Act showed high positive activity on the Gli-BS reporter, transcriptional activity that is repressed by the Gli^ZnF. Gli^ZnF alone showed no positive activity on the Gli-BS reporter. TCF^HMG had no transcriptional activity. (B-F) TCF mediated loss of Pax7 was dependent on Gli activity. (B) Twenty-four hours PE, TCF^DN alone caused loss of Pax7 expression. (C,D) Co-electroporation of TCF^DN together with Gli^ZnF totally rescued Pax7 expression. (E,F) Co-electroporation with Gli3 also rescued Pax7 expression. (G-K) Co-electroporation experiments showed partial rescue of ventral gene expression. (G) Twenty-four hours PE, TCF^DN alone induce ectopic Olig2- (green) and Nkx2.2- (red) expressing cells. (H,I) Co-electroporation of TCF^DN and Gli^ZnF resulted in the rescue of Olig2 and Nkx2.2 expression, although few ectopic Olig2+ cells still appeared. (J,K) Co-electroporation of Gli3 resulted in the rescue of Olig2 and Nkx2.2 expression. (L-Q) Twenty four hours PE of Gli3, expression of dorsal progenitor proteins Pax7 (L) and Pax6 (M) was not modified. GFP as a reporter of transgene expression (N). Expression of ventral Olig2 (O) and Nkx2.2 (P) was reduced in cells expressing the transgene (Q).

• Supplemental Figure S3 -

  Fig. S3. Analysis of the human Gli3 locus and characterization of HCNRs. Sequence alignments of the genomic interval containing the human GLI3 locus, with orthologous counterparts from representative members of the rodents, birds, amphibian and fish lineages. Conserved coding sequences are depicted in blue and conserved non-coding sequences in pink. Arrow indicates the length of the GLI3 gene and the direction of transcription. TCF-binding sites are depicted in green. Identification of consensus TCF-binding sites within the human GLI3 locus. Comparison of HCNR1-R4 sequences from the human locus with other vertebrates. All vertebrate species examined contained the core consensus TCF-biding site (green). Fugu sequence is not annotated on region R1, therefore comparison does not appear in the figure.